US2020071711A1PendingUtilityA1
Modular construction of synthetic gene circuits in mammalian cells using mathematical modeling of closed loop transcriptional repressors
Est. expiryApr 30, 2034(~7.8 yrs left)· nominal 20-yr term from priority
G16B 5/30C12N 2310/141C12N 2310/531C12Q 1/6888C12N 15/64C12N 15/635C12N 15/113C12N 15/63C12N 15/85C12N 15/907C12Q 2600/158C12N 2810/40C12Q 2600/178C12N 15/79C12N 2830/005C12N 2310/14
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Claims
Abstract
We hereby disclose and claim a mathematically based system for design and implementation synthetic biology genetic circuitry architecture which uses genetic expression circuits which are linked together to achieve a therapeutic or diagnostic effect. The genetic architecture that enables the reversible and tunable desired gene product output is achieved by genetic control elements upstream and downstream of a promoter region. Reversible and tunable control is achieved through the computational design and pairing of transcriptional repression elements with their cognate transcriptional promoters to enable a broad range of therapeutic and diagnostic applications.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A synthetic gene circuit system under mutual repression control comprising:
A plurality of upstream activation binding sites in operable association with a promoter with upstream and downstream transcriptional promoters. A first genetic control element operably configured to repress genetic transcription of a second genetic control element, A second genetic control element operably configured to repress transcription of a first genetic control element, Wherein said first genetic control element controls a first transcriptional repression element operably associated with a first transcriptional promoter and Wherein said second genetic control element controls a second transcriptional repression element operably associated with a second transcriptional promoter Wherein said first genetic control element is activated by the presence of a first microRNA to produce a first gene product with micro-RNA mediated post transcriptional regulation and Wherein said second genetic control element is activated by the presence of a second microRNA to produce a second gene product with micro-RNA mediated post transcriptional regulation.
2 . The system of claim 1 wherein production of the first gene product is within the parameters of:
d
B
dt
=
β
2
1
+
(
A
k
)
n
+
β
1
-
γ
B
=
0
and,
production of the second gene product is within the parameters of:
d
A
dt
=
β
2
1
+
(
B
k
)
n
+
β
1
-
γ
A
=
0
Wherein:
β 1 is the leakage production rate
β 2 is the maximum production rate
k is the input concentration at inhibition ratio of 50% and
n is the Hill coefficiency.
3 . The synthetic gene circuit system under mutual repression control of claim 2 wherein said first microRNA and second microRNA are exogenous to the synthetic gene circuit system.
4 . The synthetic gene circuit system under mutual repression control of claim 2 wherein said first microRNA and second microRNA are endogenous to the synthetic gene circuit system.
5 . The synthetic gene circuit system under mutual repression control of claim 2 wherein said first microRNA and second microRNA are shRNA.
6 . The synthetic gene circuit system under mutual repression control of claim 4 wherein the first microRNA is miR21.
7 . The synthetic gene circuit system under mutual repression control of claim 4 wherein the second microRNA is selected from the group consisting of miR18a, miR191, and miR19ab.
8 . The synthetic gene circuit system under mutual repression control of claim 2 additionally comprising ‘or’ logic configuration operationally configured to produce said first gene product while simultaneously inhibiting production of said second gene product.
9 . The synthetic gene circuit system under mutual repression control of claim 8 additionally comprising ‘or’ logic configuration operationally configured to produce said second gene product while simultaneously inhibiting production of said first gene product.
10 . A synthetic gene circuit system under mutual repression control comprising:
A plurality of upstream activation binding sites in operable association with a promoter with upstream and downstream transcriptional promoters. A first genetic control element operably configured to repress genetic transcription of a second genetic control element, A second genetic control element operably configured to repress transcription of a first genetic control element, Wherein said first genetic control element controls a first transcriptional repression element operably associated with a first transcriptional promoter and Wherein said second genetic control element controls a second transcriptional repression element operably associated with a second transcriptional promoter Wherein said first genetic control element is activated by the presence of a first microRNA a1 operationally configured to additional microRNA a2 to produce a first gene product with micro-RNA mediated post transcriptional regulation and Wherein said second genetic control element is activated by the presence of a second microRNA b1 operationally configured to additional micro RNA b2 to produce a second gene product with micro-RNA mediated post transcriptional regulation wherein production of the first gene product is within the parameters of:
d
B
dt
=
β
2
1
+
(
A
k
)
n
+
β
1
-
γ
B
=
0
and,
production of the second gene product is within the parameters of:
d
A
dt
=
β
2
1
+
(
B
k
)
n
+
β
1
-
γ
A
=
0
Wherein:
β 1 is the leakage production rate
β 2 is the maximum production rate
k is the input concentration at inhibition ratio of 50% and
n is the Hill coefficiency.
11 . The synthetic gene circuit system under mutual repression control of claim 10 additionally comprising logic configuration operationally configured to produce said first gene product in the presence of microRNA b1 or microRNA b2 and absence of microRNA a1 or microRNA a2 while simultaneously inhibiting production of said second gene product.
12 . The synthetic gene circuit system under mutual repression control of claim 10 additionally comprising logic configuration operationally configured to produce said second gene product in the presence of microRNA a1 or microRNA a2 and absence of microRNA b1 or microRNA b2 while simultaneously inhibiting production of said first gene product.
13 . The synthetic gene circuit system under mutual repression control of claim 10 additionally comprising:
A third genetic control element operably configured to repress transcription of a first genetic control element,
Wherein said first genetic control element controls a first transcriptional repression element operably associated with a first transcriptional promoter and
Wherein said second genetic control element controls a second transcriptional repression element operably associated with a second transcriptional promoter
Wherein said first genetic control element is activated by the presence of a first microRNA a1 operationally configured to additional microRNA a2 to produce a first gene product with micro-RNA mediated post transcriptional regulation and
Wherein said second genetic control element is activated by the presence of a second microRNA b1 operationally configured to additional micro RNA b2 to produce a second gene product with micro-RNA mediated post transcriptional regulation
Wherein said second genetic control element is additionally activated by the presence of a third microRNA c1 operationally configured to additional micro RNA c2 to produce a second gene product with micro-RNA mediated post transcriptional regulation,
Wherein production of the first gene product is within the parameters of:
d
B
dt
=
β
2
1
+
(
A
k
)
n
+
β
1
-
γ
B
=
0
and,
production of the second gene product is within the parameters of:
d
A
dt
=
β
2
1
+
(
B
k
)
n
+
β
1
-
γ
A
=
0
Wherein:
β 1 is the leakage production rate
β 2 is the maximum production rate
k is the input concentration at inhibition ratio of 50% and
n is the Hill coefficiency.
14 . The synthetic gene circuit system under mutual repression control of claim 13 additionally comprising logic configuration operationally configured to produce said second gene product in the presence of microRNA a1 or microRNA a2 and microRNA c1 or c2 and absence of microRNA b 1 or b2 while simultaneously inhibiting production of said first gene product.
15 . The synthetic gene circuit system under mutual repression control of claim 13 additionally comprising logic configuration operationally configured to produce said first gene product in the presence of microRNA b1 or microRNA b2 and microRNA c1 or c2 and absence of microRNA a1 or a2 while simultaneously inhibiting production of said second gene product.
16 . A synthetic gene circuit system under mutual repression control comprising a plurality of transcriptional repressors wherein the first transcriptional repressor and the second transcriptional repressor each have a transfer function curve determined by the following equation:
d
B
dt
=
β
2
1
+
(
A
k
)
n
+
β
1
-
γ
B
=
0
wherein A represents expression level of an input transcriptional repressor under RNA interference; B represents expression level of an output transcriptional repressor under the regulation of the input transcriptional repressor; β2 represents a maximum production rate of a promoter inhibited by the output transcriptional repressor; β1 represents a leakage production rate of a promoter inhibited by the output transcriptional repressor; k represents expression level of an input transcriptional repressor at an inhibition ratio of 50%; n represents the Hill coefficient; γ represents a decay rate reflecting RNA interference; and
β1, β2, k and n are all associated with a specific promoter and a specific transcriptional repressor, and are determined by fitting the transfer function curve with expression level of output mRNA or protein regulated by a specific input transcriptional repressor and expression level of the input transcriptional repressor under different RNA interference;
wherein an appropriate transcriptional repressor group consisting of the first transcriptional repressor and the second transcriptional repressor is determined by steps:
a. fitting a first transfer function curve reflecting inhibition of the first transcriptional repressor to the second transcriptional repressor by the equation, wherein the first transcriptional repressor and the second transcriptional repressor are respectively used as the input transcriptional repressor and the output transcriptional repressor and function solution of the equation is zero, and
b. fitting a second transfer function curve reflecting inhibition of the second transcriptional repressor to the first transcriptional repressor by the equation, wherein the second transcriptional repressor and the first transcriptional repressor are respectively used as the input transcriptional repressor and the output transcriptional repressor and the function solution of the equation is zero,
c. obtaining an intersection point of the first transfer function curve and the second transfer function curve, wherein the intersection point represents a predicted equilibrium state of the synthetic expression system, and the predicted equilibrium state is for predicting the inhibiting ability of a transcriptional repressor group with specific RNA interference,
d. for transcriptional repressor group candidates, determining expression levels of a first protein A and a first protein B regulated by the transcriptional repressor group candidate under first RNA interference, and expression levels of a second protein A and a second protein B regulated by the transcriptional repressor group candidate under second RNA interference and
e. obtaining a first difference fold and a second difference fold, wherein
First difference fold=Expression level of the first protein B/Expression level of the second protein B, wherein the expression level of the first protein B is higher than the expression level of the second protein B,
Second difference fold=Expression level of the second protein A/Expression level of the first protein A, wherein the expression level of the second protein A is higher than the expression level of the first protein A,
wherein the first difference fold and/or the second difference fold more than 5 indicates the transcriptional repressor group candidate is an appropriate transcriptional repressor group.Join the waitlist — get patent alerts
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